¹H‐NMR Study of Inter‐Segmental Hydrogen Bonds in Sperm Whale and Horse Apomyoglobins

Abstract

NMR signals for HisBS N6H and HisEFS NeH protons of sperm whale and horse apomyoglobins were assigned and compared with the corresponding signals of the holoproteins in terms of pHand temperature dependence behaviors of their shifts and line widths in order to gain insight into structural difference between the apoproteins and the holoproteins. Since these protons are involved in internal hydrogen bonds at the interfaces between the B helix and the GH corner and between the EF corner and the H helix, local structures of the interfaces in these proteins have been inferred from the analyses of these signals. A large difference in the line width of HisEF5 NeH proton signal between the apoproteins and the holoproteins strongly suggested that a sizable structural alteration is induced in the EF-H interface by the removal of heme. However, the results for HisBS N6H proton resonance indicated the absence of a significant structural alteration in the B-GH interface by heme extraction. These results are consistent with the data obtained from mutation [Hughson, F. M.

Fe-Heme Conformations in Ferric Myoglobin

“…From top to bottom, the former three spectra follow the pH dependence of Mb ϩ at T ϭ 293 K, the latter four spectra the temperature dependence at pH ϭ 11.3. Increasing the pH induces discontinuous protein changes between different conformations (Yamamoto, 1997), distinct from substates (Frauenfelder et al, 1988). Because the visible absorption spectra are sensitive to only two Fe-heme states, we assume that these subtle changes pertain to the axial symmetry of the Fe-heme, due to proton titration of the distal and proximal histidines.…”

Solvent Effects on Horse Apomyoglobin Dynamics

“…As a consequence, when apoMb is subjected to solvent perturbation, the compactness of this domain (revealed by the limiting Trp fluorescence anisotropy) appears largely preserved. Me- chanical constraints, which disrupt or connect a few stable domains, only affect the tertiary structure (12). Though the two Trp residues embedded in this domain are highly protected, both can be reached by the DCA molecules which cross this cluster during their random migration through the protein.…”

“…On the myoglobin molecule, they only lessen or increase the tensions on the hydrated β-turns (11). On the other hand, pH controls the hydrogen bonds between particular histidine residues of adjacent chains (12). The protein dynamics of myoglobin could be coupled to these structural changes.…”

“…When pH reorganizes specific tertiary contacts inside this protein (12), or organic solvents induce particular strains on β-turns (11), these perturbations can produce important changes in the compactness of the bulk protein where the Trp residues are located. In this case, the protein strains should induce differences in the electronic organization of the buried Trp (22)(23)(24) and thus modify the values of the Trp limiting anisotropy.…”

Characterization of Adducts of Ethanol Metabolites with Cytochrome c

“…The binding of EtOH to myoglobin (Mb) and apoMb supplemented with peroxide was also investigated to understand the role of the heme prosthetic group and the apoprotein. Mb is of similar size and structure to cyt c; but, unlike the latter, it has a noncovalently bound heme moiety that can be removed easily without structural collapse of the apoprotein (Yamamoto, 1997). Production of HER was determined with electron spin resonance (ESR) spectroscopy in the presence of a spin trapping agent.…”